Vision-impairing corneal scarring following injuries caused by trauma, surgery, or infection remains a major clinical problem, yet no drugs are FDA-approved with the claim of reducing light-scattering corneal haze. Clinical trials of anti-inflammatory steroids have not shown statistically significant benefit, and mitomycin C treatment carries the risk serious side effects. Cell culture studies, animal experiments, and recent clinica trials strongly indicate that connective tissue growth factor (CTGF) is the dominant scar-inducing growth factor in the cornea and in other tissues, including skin and kidney. CTGF generates corneal scar by up-regulating synthesis of irregular extracellular scar matrix and inducing transformation of quiescent keratocytes into activated fibroblasts and myofibroblasts, which combine to produce the majority of light scattering in corneal scars. Our overall goal is to understand the basic molecular regulation of corneal wound healing and apply that knowledge in the development of even more effective therapies to reduce corneal scarring. In this new proposal, we propose three specific aims that further expand our knowledge base of the CTGF system in corneal wound healing and translate that information into new clinical therapies that eliminate/reduce corneal scarring. Specific Aim #1 will further define the key roles of CTGF in regulating corneal scarring using three approaches. We will identify the cells in the cornea that synthesize CTGF during wound healing using CTGF promoter-GFP transgenic mice and Q-RT-PCR analysis of samples of epithelial cells, stromal cells and endothelial cells. We will determine the maximum reduction in corneal scarring that can be achieved by eliminating CTGF synthesis in wounded corneas using a new CTGF conditional knockout mouse strain that we will develop that stops synthesis of CTGF after exposure to tamoxifen (CTGFfloxed/floxed CreCAG-cre/Esr/+). We will also use this unique CTGF conditional knockout mouse strain assess if other growth factor systems may partially compensate for elimination of CTGF synthesis and contribute to scarring. Epigenetic regulation of corneal wound healing is a relatively unexplored area. Specific Aim #2 will investigate changes in miRNAs patterns using miRNA microarrays. We also will assess the effects two classes of epigenetic drugs, microRNAs (miRNA) and histone deacetylase inhibitors (HDI), on synthesis of CTGF in corneal epithelial cells, fibroblasts and endothelial cells. Finally, we hypothesize that maximum reduction of corneal scarring will be achieved using a combination of gene targeted and broad based epigenetic knockdown. Therefore, Specific Aim #3 will evaluate antiscarring effects of a combination of CTGF-ASOs and epigenetic treatments using the HDI, SAHA, and microRNAs, mir26a, and mir18a that have been reported to reduce synthesis of CTGF in other cell types. PUBLIC HEALTH RELEVANCE: Vision-impairing corneal scarring following injuries caused by trauma, surgery, or infection remains a major clinical problem, yet no drugs effectively reduce corneal scarring without the risk of serious side effects.(1),(2),(3) Our research identified two growth factors that are the major regulators of scar formation in the cornea, transforming growth factor beta (TGFb), and its downstream mediator, connective tissue growth factor (CTGF).(4) This grant will continue our basic research to understand how the CTGF system functions during corneal wound healing using CTGF conditional knockout mice and cell culture systems and translate that knowledge into novel drug therapies that reduce corneal scarring without producing serious, long-term side effects using both specific gene-targeted therapies and broad-based epigenetic therapies.